Microbial group specific uptake of inorganic phosphate and ATP at station ALOHA: kinetics, effect of light and
response to rapid changes in N:P availability
OSM 2014
Session 049
Poster # 2435
Solange Duhamel1,2, Karin M. Björkman1, Joseph K. Doggett1, David M. Karl1
1Department
of Oceanography, Center for Microbial Oceanography: Research and Education (C-MORE), University of Hawaii, 1950 East West Road, Honolulu, Hawaii 96822, USA
2Present address: Lamont-Doherty Earth Observatory (LDEO), Division of Biology and Paleo Environment, Columbia University, PO Box 1000, 61 Route 9W, Palisades, New York 10964, USA
Method for group specific P uptake rates
33PO
4
Phosphorus (P) is an essential element for all living organisms1. Marine microbes
generally prefer dissolved inorganic P (Pi) in the form of PO43−, but Pi availability is
frequently in low, or limiting supply relative to biological demand in aquatic
ecosystems2. The most abundant microbes in the wide oligotrophic ocean,
Prochlorococcus (PRO), Synechococcus (SYN) and non-pigmented bacteria (NPB)
are osmotrophic and rely on a mixture of Pi and dissolved organic P (DOP)
compounds to support their nutritional needs3. These groups may be in direct
competition, as well as with larger phytoplankton, when essential resources are in
short supply. We aim to better assess the P uptake strategies in important groups of
microbes in the North Pacific subtropical gyre (NPSG).
or 33P-ATP
Red fluorescence (A.U.)
Seawater
sample
Flow cytometry
3
Incubation
1
2
104
104
Beads
103
102
Prochlorococcus
101
102
101
Beads
103
Non-pigmented
Bacteria
102
101
101
103
102
103
FSC PAR (A.U.)
Flow cytometry
cell sorting
Hypotheses
1. Concentration dependent uptake of
Pi and ATP in microbial populations
4
The BD Influx - high-speed sorter and analyzer
1. The relative success of specific groups of microorganisms may be related to their P uptake kinetic
response characteristics
2. Light stimulates Pi and DOP uptake by Prochlorococcus
3. Synechococcus may be a major player in the NPSG P cycle when nitrogen limitation is relieved
Liquid scintillation
radioactivity counting
5
2. Effect of light on Pi and ATP uptake
Pi uptake rate (nmol
0.00 0.05 0.10 0.15 0.20
0
Depth (m)
L-1h-1)
0.25
50
L:D
Light uptake
Dark uptake
100
150
200
0.0
0.5
1.0
1.5
2.0
Ø  PRO uptake of both Pi and ATP
were higher when the samples
were incubated under ambient
light
Ø  while there was no significant
difference in P-assimilation by
non-pigmented picoplankton
between light and dark incubated
samples.
L:D Pi uptake rate ratio
3. Microbial response to enhanced P cycling
0
4
4
3
3
2
2
1
1
0
50 100 150 200 250
Pi (n+s) (nmol L-1)
Organic
phosphorus
0
NPB
20
40
ATP (n+s) (nmol L-1)
0
0
50 100 150 200 250
Pi (n+s) (nmol L-1)
16
4
12
3
8
2
4
1
0
60
0
20
40
ATP (n+s) (nmol L-1)
0
60
Approach: To induce P depletion, surface NPSG water was amended with two different
inorganic N additions to yield N:P ratios of NP32 and NP50, and incubated over 6 days
Initial conditions @ ALOHA: total dissolved N (~6 µM) and P (~200-300 nM) = N:P ~21
•  N:P = 32
Typically measured in the low-P Mediterranean Sea and
•  N:P = 50
Western North Atlantic ocean
After 6 days of incubation in NP50:
Ø  NPB increased their group-specific rates of Pi uptake by 4 to 6 times compared to
control, while ATP uptake rates stayed the same
Ø  SYN abundance and cell-specific Pi and ATP uptake rates greatly increased (to 56X,
12X and 8X their initial abundance, cell-specific Pi and ATP uptake rates, respectively),
and SYN group-specific rates of Pi and ATP uptake reached rates similar to NPB.
Ø  Two populations of Synechococcus could be distinguished
Examples of density plots of phycoerythrin (y-axis) vs. chlorophyll (x-axis) fluorescence (arbitrary units (A.U.)) obtained
by flow cytometry after 6 days of incubation for the control (E), NP32 (F) and NP50 (G) treatments. The single
population of Synechococcus (E) can be compared to the two populations of Synechococcus (S1 and S2) identified in
the N-amended treatments (F and G). Calibration beads (Beads, 1-µm diameter) were used for reference.
L:D γ ATP uptake ratio
Inorganic
phosphorus
PRO
L:D Pi uptake ratio
Light dependence of P-utilization: group-specific response Examples of response in Pi and ATP uptake rates (nmol L-1 d-1) to increasing concentrations of Pi or ATP.
Per cell and
per population P
uptake rates
Light dependence of P-utilization: bulk rates Microbial group and cell specific P uptake rates of Pi and γ-P-ATP at ambient concentrations; and the calculated kinetic
parameters Vmax and Km for incubations containing additional substrate.
Routine additions were 0, 10, 25, 50, 75, and 150 nmol L-1 Pi, and 0, 2, 5, 10, 25, and 50 nmol L-1 ATP (equivalent to
three times that concentration in terms of P additions, to achieve the same loading as in the Pi amended experiments).
Inorganic
phosphorus
Station locations
Stop incubation (cold chase)
Green fluorescence (A.U.)
Introduction
Organic
phosphorus
Conclusions and new hypotheses to be tested
Ø  PRO and NPB were in close competition in terms of Pi acquisition, whereas P uptake
from ATP could be attributed to NPB. This apparent resource partitioning may be a niche
separating strategy and an important factor in the successful co-existence within the
oligotrophic upper ocean of the NPSG.
Ø  Kinetic response to Pi was small, indicating that PRO and NPB were close to their Vmax
Ø  PRO had three times the cell specific Pi uptake rate of NPB, at ambient
concentrations, but when adjusted to cells L−1 the rates were similar, and these two
groups were equally competitive for Pi.
Ø  PRO had Km values 5 to 10 times greater than NPB
Ø  Yet, in N-amended samples, SYN cell abundance and cell-specific P uptake rates were
greatly enhanced and SYN group-specific rates were comparable to those of NPB. Two
new populations of SYN could be distinguished by flow cytometry.
u Distinct populations of SYN may be better suited to thrive in low-P environments
Ø  PRO uptake of Pi and ATP was higher in samples incubated under ambient light and the
L:D ATP uptake ratio decrease with increasing ATP additions.
u The effect of light on P uptake may be larger in P substrate limited conditions
Acknowledgments: Many thanks to the captains and crew of the R/V Kilo Moana, R/V Kaimikai-O-Kanaloa
and R/V Melville. We thank the chief scientists of the HOT, KM1016, OPEREX and BiG RAPA cruises for
accommodating our needs on these cruises. We thank N. Hakoda, M. Segura, J. C. Jennings, Jr. and S.
Curless for assistance with sample analyses. Funds for this work were provided by the Gordon and Betty
Moore Foundation’s Marine Microbiology Initiative (D.M.K.) and the National Science Foundation (D.M.K.,
EF0424599; M.J. Church, OCE-0926766).
References
1. Karl, 2000, Nature
2. Dyhrman et al, 2007, Oceanography
3. Thingstad et al, 1993, MEPS
Corresponding papers
²  Duhamel et al 2014. Microbial response to enhanced phosphorus cycling in the North Pacific Subtropical
Gyre. MEPS doi: 10.3354/meps10757
²  Duhamel et al 2012. Light dependence of phosphorus uptake by microorganisms in the North and South
Pacific subtropical gyres. AME 67: 225-238, doi: 10.3354/ame01593
²  Björkman et al 2012. Microbial group specific uptake kinetics of inorganic phosphate and adenosine-5’triphosphate (ATP) in the North Pacific Subtropical Gyre. Frontiers in Microbiology 3(189):1-17
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